Access to Titan Krios Cryo-TEM
The Titan Krios cryo-STEM ("Krios") is a high-throughput instrument capable of autonomously collecting large volumes of data. It is, however, a costly instrument to operate and in high demand by research groups throughout the region and beyond.
To maximize the serviceable user time on the Krios, FEMR staff will perform operations for most users. This includes grid clipping, loading, unloading, transfers, microscope alignments, low-dose imaging, data collection, and troubleshooting. Following extensive training and approval by FEMR staff, some users may be allowed to perform some tasks independently.听
The FEMR has a screening policy and quality checks before聽any sample may be聽loaded into the Krios.
The first step to accessing the Krios is, as for any project, to submit the Project Form and attend the Assessment Interview. Once the project is deemed appropriate for the Krios, the submission of samples聽will follow the cryo grid quality control procedure and submission of the聽Project Form for Cryo-EM.
Procedure for Cryo Grid Quality Control
No samples may be loaded into the Krios without being聽previously screened by negative-stain TEM聽and cryo-EM.
Negative-stain TEM聽is the聽conventional method of staining samples on a TEM grid聽with uranyl acetate or uranyl formate and imaging by the FEI Tecnai Spirit 120 kV TEM or Tecnai G2 F20 cryo-TEM.
Cryo-EM screening will involve vitrification of the sample by聽the FEI Vitrobot Mk IV and imaging聽in the FEI Tecnai G2 F20 200 kV or Tecnai Spirit 120 kV cryo-TEM (equipped with a cryo-box).
Requirements for imaging samples by the Krios:
- Low-magnification images showing high-quality ice of appropriate thickness can be obtained for聽the sample.听
- High-magnification cryoEM images showing visible particle distribution, absence of aggregation and contamination, and good ice.
- A full cryoEM grid map (Atlas) using SerialEM.
- Once items 1, 2, and 3 are acquired, submit the completed聽Project Form-CryoEM.听
- The most efficient way to ensure successful data collection聽on聽the Krios is to validate the Vitrobot blotting and concentration conditions by screening the grids with either the FEI Tecnai G2 F20 TEM or Tecnai G2 Spirit TEM聽and then freezing聽ten new grids using the same conditions. Again, screen one聽or two聽of these ten grids on either TEM and if these grids have good ice and particle distribution, load a set of four grids into the Krios. If none of the four grids is suitable, you can load another four grids for a charge of $500.听
Following these steps will ensure the聽cryo grids have聽proper ice thickness, good particle distribution, and abundant usable area on a single grid, thus, making good use of valuable microscope time.
Once the聽Project Form for Cryo-EM has been processed, your name and number聽of days on the Krios will be聽uploaded to the聽Krios Waiting List.听No details of聽the project will appear on this list.听罢测辫颈肠补濒濒测,听projects are added on a first-come, first-served basis.听Any request for placing a project on the list using a different criterion听(颈.别.,听manuscript revision with a deadline, grant application, etc.) will be assessed聽by the Krios Screening Committee听补苍诲听approved only if 谤别补蝉辞苍补产濒别听justification is provided.听
External clients who wish to ship cryo grids to 捆绑SM社区 should ensure they arrive within one to five聽business days before their session on the Krios. It is, however, acceptable for users to bring聽the聽grids, but they should be ready to load the samples by 9:00 am on the聽first day聽of their scheduled session.